bioRxiv preprint doi: https://doi.org/10.1101/2021.02.23.432581; this version posted February 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Spatially-Resolved Proteomic Analysis of the Lens Extracellular Diffusion Barrier Zhen Wang, and Kevin L Schey* Department of Biochemistry, Vanderbilt University, Nashville, TN 37232 Key words: lens fiber cells, extracellular diffusion barrier, laser capture microdissection, quantitative mass spectrometry * To whom correspondence should be addressed Current address: Mass Spectrometry Research Center Vanderbilt University 465 21st Ave. So., Suite 9160 MRB III Nashville, TN 37232-8575 E-mail: [email protected] Phone: 615.936.6861 Fax: 615.343.8372 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.23.432581; this version posted February 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Purpose: The presence of a physical barrier to molecular diffusion through lenticular extracellular space has been repeatedly detected in multiple species. This extracellular diffusion barrier has been proposed to restrict the movement of solutes into the lens and to direct nutrients into the lens core via the sutures at both poles. The purpose of this study is to characterize the molecular components that could contribute to the formation of this barrier. Methods: Three distinct regions in the bovine lens cortex were captured by laser capture microdissection guided by dye penetration. Proteins were digested by endoproteinase Lys C and trypsin. Mass spectrometry-based quantitative proteomic analysis followed by gene ontology (GO) and protein-protein interaction network analysis was performed. Results: Dye penetration showed that lens fiber cells first shrink the extracellular spaces of the broad sides of fiber cells followed by closure of the extracellular space between narrow sides at normalized lens distance (r/a) of 0.9. Accompanying the closure of extracellular space of the broad sides, dramatic proteomic changes were detected including up-regulation of several cell junctional proteins. AQP0 and its interacting partners ERM proteins were among a few proteins that were upregulated accompanying the closure of extracellular space of the narrow sides suggesting a particularly important role for the major lens membrane protein AQP0 in controlling the narrowing of the extracellular spaces between lens fiber cells. The results also provided important information related to biological processes that occur during fiber cell differentiation such as organelle degradation, cytoskeletal remodeling and GSH synthesis. Conclusions: The formation of lens extracellular diffusion barrier is accompanied by significant membrane and cytoskeletal protein remodeling. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.23.432581; this version posted February 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction To achieve its normal function as an optical element and to maintain its transparency, the lens has evolved a unique structure that lacks light-scattering elements including blood vessels and cellular organelles (1). In addition, the terminally differentiated fiber cells are closely packed with the extracellular space smaller than the wavelength of the light to reduce light scattering (1). In the absence of a blood supply, this large tissue cannot rely on simple diffusion to deliver nutrients to the center of the lens. It has been demonstrated that the lens establishes a microcirculation system to deliver nutrients, remove wastes, maintain ionic homeostasis, and to control the volume of the fiber cells (1-3, 4). Based on the microcirculation system model, a circulating current, carried primarily by Na+ ions, directs nutrients extracellularly to the center of the lens at both poles via the sutures. Na+ ions along with water and metabolites in the center of the lens diffuse toward the lens equator intercellularly, i.e. from cell to cell across cell membranes, and flow toward the lens surface through gap junctions. The formation of the microcirculation system relies on spatially distinct distributions of ion channels and transporters including highly concentrated activity of sodium-potassium pumps in the anterior epithelial cells (5-6) and a high gap junction coupling conductance at the equator (7). The circulating current at the surface of the lens has been confirmed experimentally (8). Magnetic resonance imaging (MRI) has allowed visualization of fluid fluxes of heavy water (D2O) that are directed into the lens at the poles and then move circumferentially toward the equator in the lens cortex (9). This study also identified a zone of restricted extracellular space diffusion (9). A physical barrier to extracellular space diffusion has been reported in lenses from different species (10-11) which is consistent with the observed reduction of extracellular space between fiber cells to avoid light scattering that has been confirmed by electron microscopy (12). This extracellular diffusion barrier has been proposed to restrict the movement of solutes into the lens and acts to direct nutrients into the lens core via the suture at both poles (9). The molecular mechanism that controls closing of the extracellular space and formation of the extracellular diffusion barrier has not been studied. Tight junctions are normally believed to control paracellular permeability and provide a diffusion barrier (13). Occluded zones similar to tight junctions were described between lens fiber cells in an early EM study (12). Several well- known tight junction proteins can be detected in lens fiber cells such as ZO-1, junction adhesion molecule 3 (JAM3) and coxsackie and adenovirus receptor (Cxadr) (14, 15). However, due to bioRxiv preprint doi: https://doi.org/10.1101/2021.02.23.432581; this version posted February 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. lack of core proteins of tight junction occludins or claudins, tight junctions were reported to be absent in lens fiber cells (14, 15, 16). In the absence of claudins, lens fiber cells express LIM2; a member of the PMP-22/EMP/MP20/Claudin Family that shares a tetraspanin topology and the WGLWCC signature sequence in the first extracellular loop (17). The precise function of LIM2 is unknown, but an adhesive function (18) and a role in fiber cell junction formation and organization has been reported (19). Another unique structure present in lens fiber cells is the square array junction (20-22). AQP0 and its cleavage products were found to be enriched in square array junctions (20, 21). Previous electron microscopy data suggested that the reduction of lenticular intercellular space was correlated with the formation of square array and membrane undulation (20, 21). Thus, square arrays might potentially drive the formation of complicated membrane interdigitations and serve to maintain an extremely narrow extracellular space (20, 22). In addition, it has been reported that gap junction plaques on the broad side of the outer fiber cells could restrict penetration of larger solutes through extracellular space (9). The purpose of this study was to investigate proteins that could regulate and control the closure of lens extracellular spaces. Previous studies mainly relied on immunohistochemistry, and confocal and electron microscopy (10, 11, 20, 22). Mass spectrometry-based quantitative proteomic analysis is a powerful discovery approach to examine global proteomic dynamics. We hypothesize that an in-depth spatially resolved proteomic study will reveal proteins and pathways that could be involved in controlling extracellular space and diffusion. In this paper, a dye penetration experiment defined three distinct regions within less than 1.5 mm distant from the lens capsule to inner cortex in the equatorial region of bovine lenses. These three regions were then isolated by laser capture microdissection and proteome changes across three regions were studied with high-throughput quantitative mass spectrometry. Our results showed that the extracellular diffusion barrier is formed in a stepwise manner accompanied with dramatic changes in fiber cell junctional proteins, membrane and cytoskeletal proteins. Materials and Methods Dye penetration and confocal imaging Bovine lenses were extracted from fresh cow eyes (Light Hill Meats, Lynnville, TN) and incubated in M199 medium (Sigma, St. Louis, MO) that contained 1mg/ml Texas Red-dextran (10,000 MW, Lysine Fixable, Thermo Fisher Scientific, Rockford, IL) at 37oC for 6-24 hr. All bioRxiv preprint doi: https://doi.org/10.1101/2021.02.23.432581; this version posted February 23, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. lenses used in this study were from cows of about 2-years-old. After incubation, lenses were rinsed with M199 media and fixed in 50 mL of 2% paraformaldehyde containing 0.01% glutaraldehyde for 4 days. The lenses were washed three times with PBS for 20 min and then cryoprotected with 10% sucrose in PBS for one